Sustained Inhibition of Whole-Blood Clot Procoagulant Activity by Inhibition of Thrombus-Associated Factor Xa

Abstract

Progression of arterial thrombosis partly depends on thrombus-associated thrombin and activated factor X (Xa) activity. However, whether Xa or thrombin is the most appropriate target for inhibition of recurrent thrombosis is unknown. This study was designed to determine whether inhibition of Xa results in more sustained attenuation of thrombus-associated precoagulant activity than does inhibition of thrombin. Clots prepared ex vivo from human whole blood and pathological arterial thrombi from patients were preincubated in citrated plasma containing no inhibitor, 0.5 to 1 U/mL heparin, 0.5 to 1 mumol/L hirudin, 5 to 10 mumol/L tick anticoagulant peptide (TAP), 0.15 to 3 mumol/L tissue factor pathway inhibitor (TFPI), or a combination of 1 mumol/L hirudin and 10 mumol/L TAP for 2 hours. After preincubation the clots were removed from first-stage plasma, extensively washed in phosphate-buffered saline, and added to nonanticoagulated whole blood. Clots preincubated in plasma without inhibitors induced marked activation of the coagulation system in whole blood, as characterized by greater increases in the concentration of fibrinopeptide A (FPA) over 7 minutes than in blood without added clots (1522 +/- 568 compared with 117 +/- 170 ng/mL, P < .01). Preincubation of clots with heparin or hirudin did not attenuate the increases in FPA in wholeblood. In contrast, compared with incubation without an inhibitor, preincubation of clots with TAP or TFPI markedly attenuated the increases in FPA when clots were added to whole blood (551 +/- 316 and 508 +/- 208 ng/mL, respectively, P < .01). Similar results were obtained with arterial thrombi from patients. Inhibition of Xa but not of thrombin results in sustained attenuation of thrombus-associated procoagulant activity. Uninhibited thrombus-associated Xa activity may account for the increases in thrombin activity that are commonly observed in clinical trials after discontinuation of thrombin inhibitors.

Prolonged Activation of Prothrombin on the Vascular Wall After Arterial Injury

“…Our previous findings document the observation that conditions which induce more extensive mural vascular injury and platelet-rich thrombosis (ie, high shear, electrical injury) cause considerably more Xa/Va activity to be associated with the thrombus, leading to marked local generation of thrombin. 4,8,10,11 Persistence of Bound Xa/Va Activity After Arterial Injury Data from previous experimental and clinical studies are consistent with our observation that Xa/Va activity persists for long periods of time after initial arterial injury. For example, using assays similar to those we used, Barry et al 19 have shown that a 2-hour infusion of hirudin attenuates arterial wallassociated thrombin activity at 24 hours but that bound thrombin activity increases by 48 hours.…”

“…These results suggest that the extent of Xa/Va activity after injury in this preparation is substantially less than that we have previously observed associated with arterial thrombi formed in vivo; the thrombi induce clotting in less than 15 minutes under similar conditions. 8,10 However, the importance of bound Xa/Va activity should not be underestimated, because the thrombin elaborated may markedly potentiate thrombosis by activating the coagulation cofactors V and VIII and by activating platelets. 3,7 In our preparation of balloon-induced aortic injury, mural thrombus was not observed, but platelet and fibrin deposition occurred.…”

“…We hypothesized that the Xa/Va complex expressed by platelets that bind to the vascular wall after injury may be an important determinant of thrombin elaboration. 8,10,11 Accordingly, the objectives of this study were to characterize the functional activity of Xa/Va and thrombin associated with the vascular wall after balloon-induced injury to the rabbit aorta and the extent to which the activity of these factors contributes to procoagulant activity over time. In addition, we characterized the extent to which aspirin and heparin attenuate vascular wall-associated procoagulant activity after injury.…”

Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

“…By the addition of the factor Xa recognition sequence, the fusion protein inhibited thrombin only in the presence of factor Xa. Because this factor is part of the activated clotting system and is an important determinant of the procoagulant activity of whole blood clots and arterial thrombi, 15 the designed fusion protein represents an anticoagulant that promises to be preferentially active at the site where it is needed.…”

“…First, factor Xa cleaves at the C-terminus of its recognition sequence (Ile-Glu-Gly-Arg) and thus liberates the free amino-terminus of hirudin. Second, factor Xa is a major part of the activated coagulation system at the site of arterial clots 15 and may therefore allow a preferential liberation of functional hirudin at the clot. Without an activated coagulation system, the fusion protein would be inert.…”